Mosquito-borne diseases remain significant causes of morbidity and mortality throughout many tropical regions of the world and impose a tremendous burden on human populations. The outbreak of West Nile virus in the United States within the last 3 to 4 years demonstrates that this country is not immune to the threat of mosquito-borne pathogens. It is critical that we have a better understanding of the molecular basis of pathogen transmission by Anopheles, Aedes, and Culex spp. Genome sequence information is now available for at least one vector species (An. gambiae) and a number of arthropod borne pathogens. The sequence of the Ae. aegypti genome will be available soon. We need the molecular tools for developing a more complete understanding of mosquito-borne disease transmission. We expect that alphavirus transducing systems (ATS's) will be essential tools in the vector biologist's tool kit that will facilitate research in vector biology, especially research requiring expression of exogenous and endogenous genes in the vector and RNA silencing for functional analyses of genes. In this proposal, we will use genetically stable Sindbis-based ATS's that express a green fluorescent protein marker to facilitate pathogenesis studies in mosquitoes and help us map determinants of ATS injection in Ae. aegypti. We also will assess the role of these determinants in the infection of other mosquito species. We will use the knowledge we have gained during the previous granting period to reengineer ATS's to increase their utility for vector research. We also will develop novel ATS's to target vectors and vector organs and tissues that are currently not permissive for ATS infection and design new ATS's to facilitate functional analyses of mosquito genes. Knowledge gained from this proposal should aid in the understanding of vector-pathogen interactions, result in more efficient and safer ATS's, and point the way towards designing general strategies for development of ATS's for use in other vector species. The specific aims of this proposal are as follows: 1. Use ATS's to define specific alphavirus determinants in the E2 glycoprotein that allow enhanced mosquito infection by the oral route. 2. Develop ATS's with a novel "gateway" peptide ligand in the E2 glycoprotein to facilitate gene expression in other medically important arthropod vector species. 3. Understand and improve ATS's induction of RNA silencing in mosquitoes.